APTO-253 Interaction with G-Quadruplex DNA Is Linked to Inhibition of c-Myc Expression, Induction of DNA Damage, and Generation of Synthetic Lethality in Cells with BRCA1/2 Impairment

APTO-253 is a Phase 1 clinical stage small molecule that selectively kills acute myeloid leukemia (AML) cells in the absence of toxicity to normal peripheral blood mononuclear (PBMCs) and bone marrow cells in various animal species and humans. Previously, APTO-253 was reported to exhibit concentration and time dependent inhibition of c-MYC oncogene expression in AML cells and consequently to induce G0/G1 cell cycle arrest and apoptosis, but not affect normal PBMCs (58^th ASH abstract #1716). Current mechanistic studies demonstrated that APTO-253 modulates c-MYC at the transcriptional level by decreasing acetylated H3K27 at its promoters and additionally by destabilizing c-MYC mRNA. In addition, differential gene expression analysis of RNA-seq and reverse phase protein array (RPPA) data highlighted a role for c-MYC in the mechanism of APTO-253 (GO terms - Down regulated by c-MYC p-value 6E^-26, Gene promoters bound by c-MYC p-value 4.2 E^-10, ChIP targets of c-MYC p-value 3.3E^-8). Furthermore, from the RPPA data we observed an increase in p-Chk1, p-Chk2, γH2Ax, and total p53 and E2F1, all of which are indicative of activation of DNA damage response pathways. This was accompanied by elevated levels of XBP1, GRP78, and p-p38 that point towards cellular stress response signaling (GO term Regulation of Cell Stress, p-value 1.89E^-8). Subsequent work demonstrated that APTO-253 induced DNA damage as documented by gH2AX foci formation and the neutral COMET assay, and exhibited synthetic lethality in cells with BRCA1/2 mutations that have impaired ability to repair DNA damage by homologous recombination (abstract submitted to 59^th ASH annual meeting). This amalgam of data, coupled with structural informatics analyses, suggested a mechanism of action involving the binding and/or stabilization of G-quadruplex (G4) DNA motifs. Intracellular pharmacokinetic studies revealed that APTO-253 converts from a monomer to a ferrous complex. PCR stop and modified FRET assays demonstrated that the APTO-253 ferrous complex Fe(253)₃ has activity similar to that of TMPyP4, a well-established G4 ligand, to bind/stabilize G4 structures in telomere sequences (∆T_1/2 vs concentration slope 7.7 ±2.7 and 10.67 ±1.23 for Fe(253)₃ and TMPyP4, respectively), in MYC promoter sequences (5.07 ±1.56 and 5.62 ±0.75, respectively) and in rRNA sequences (4.6 ±1.1 and 5.6 ±0.88, respectively). Although APTO-253 may participate in multiple mechanistic events, the effect of APTO-253 on c-MYC expression, cell cycle arrest and DNA damage, as well as synthetic lethality in cells with compromised DNA repair mechanisms, can be explained by the action of the Fe(253)₃ complex on G-quadruplex DNA motifs.